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[Image: see text] Spiroimines are a class of compounds produced by marine dinoflagellates with a wide range of toxicity and therapeutic potential. The smallest of the cyclic imines, portimine, is far less toxic than other known members in several animal models. Portimine has also been shown to induce apoptosis and reduce the growth of a variety of cancer cell lines at low nanomolar concentrations. In an effort to discover new spiroimines, the current study undertook a metabolomic analysis of cultures of cyclic imine-producing dinoflagellates, and a new analog of portimine was discovered in which the five-membered cyclic ether is open. Further scrutiny with human oral cavity squamous cell carcinoma (OCSCC) cell lines revealed that the open ring congener was less potent than portimine A but could still lead to the accumulation of apoptotic gene transcripts, fragment genomic DNA, and reduce cancer cell proliferation in the range of 100–200 nM.

The global need for accurate and sensitive quantitation of microcystins (MCs) persists as incidents of cyanobacterial harmful algal blooms continue to rise and recent research reveals an underestimation of the human health implications of these toxins. An optimal approach for their accurate quantitation relies on the availability of stable isotope-labeled MC standards for use in stable isotope dilution analysis (SIDA) strategies involving liquid chromatography tandem mass spectrometry (LC-MS/MS). Due to the dearth of isotopically labeled MCs, ten different 15N-enriched MCs were biosynthesized from producing cultures and fully characterized. This involved the comparative MS/MS fragmentation of natural abundance or unlabeled metabolites with their 15N-labeled congeners for improved confidence in product ion annotation. These results revealed a series of incorrect annotations described previously in the literature. In this manuscript, the biosynthesis of labeled microcystin is detailed, and their complete analytical characterization for prospective use in targeted SIDA applications, such as routine water testing is described.

An untargeted metabolomics approach was used to investigate a cultured strain of Microcystis aeruginosa (UTEX LB2386) known to be a prolific producer of a diverse class of cyanopeptides. Identification of a putative new compound with a molecular weight of 996 led to the purification and structure elucidation of this new member of the micropeptin class of cyanopeptides. Micropeptin 996 displayed potent inhibition of the serine protease enzyme chymotrypisin relative to structurally related members of this class.

Marine microorganisms have been a resource for novel therapeutic drugs for decades. In addition to anticancer drugs, the drug acyclovir, derived from a marine sponge, is FDA-approved for the treatment of human herpes simplex virus-1 infections. Most alphaviruses that are infectious to terrestrial animals and humans, such as Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV), lack efficient antiviral drugs and it is imperative to develop these remedies. To push the discovery and development of anti-alphavirus compounds forward, this study aimed to isolate and screen for potential antiviral compounds from cultured marine microbes originating from the marine environment. Compounds from marine microbes were of interest as they are prolific producers of bioactive compounds across the spectrum of human diseases and infections. Homoseongomycin, an actinobacteria isolated from a marine sponge displayed impressive activity against VEEV from a total of 76 marine bioactive products. The 50% effective concentration (EC50) for homoseongomycin was 8.6 μM for suppressing VEEV TC-83 luciferase reporter virus replication. Homoseongomycin was non-toxic up to 50 μM and partially rescued cells from VEEV induced cell death. Homoseongomycin exhibited highly efficient antiviral activity with a reduction of VEEV infectious titers by 8 log10 at 50 μM. It also inhibited EEEV replication with an EC50 of 1.2 μM. Mechanism of action studies suggest that homoseongomycin affects both early and late stages of the viral life cycle. Cells treated with 25 μM of homoseongomycin had a ~90% reduction in viral entry. In comparison, later stages showed a more robust reduction in infectious titers (6 log10) and VEEV extracellular viral RNA levels (4 log10), but a lesser impact on intracellular viral RNA levels (1.5 log10). In sum, this work demonstrates that homoseongomycin is a potential anti-VEEV and anti-EEEV compound due to its low cytotoxicity and potent antiviral activity.

Many toxic secondary metabolites used for defense are also toxic to the producing organism. One important way to circumvent toxicity is to store the toxin as an inactive precursor. Several sulfated diesters of the diarrhetic shellfish poisoning (DSP) toxin okadaic acid have been reported from cultures of various dinoflagellate species belonging to the genus Prorocentrum. It has been proposed that these sulfated diesters are a means of toxin storage within the dinoflagellate cell, and that a putative enzyme mediated two-step hydrolysis of sulfated diesters such as DTX-4 and DTX-5 initially leads to the formation of diol esters and ultimately to the release of free okadaic acid. However, only one diol ester and no sulfated diesters of DTX-1, a closely related DSP toxin, have been isolated leading some to speculate that this toxin is not stored as a sulfated diester and is processed by some other means. DSP components in organic extracts of two large scale Prorocentrum lima laboratory cultures have been investigated. In addition to the usual suite of okadaic acid esters, as well as the free acids okadaic acid and DTX-1, a group of corresponding diol- and sulfated diesters of both okadaic acid and DTX-1 have now been isolated and structurally characterized, confirming that both okadaic acid and DTX-1 are initially formed in the dinoflagellate cell as the non-toxic sulfated diesters.

We report a mass-spectrometry-based metabolomics study of a laboratory-cultured strain of Microcystis aeruginosa (UTEX LB2385), which has led to the discovery of five peptides (1–5) belonging to the microginin class of linear cyanopeptides. The structures and configurations of these peptides were determined by spectroscopic analyses and chemical derivitization. The microginin peptides described herein are the first reported derivatives containing N-methyl methionine (1, 5) and N-methyl methionine sulfoxide (2–4). The two tripeptide microginin analogues (4, 5) identified represent the smallest members of this peptide family. Their angiotensin-converting enzyme (ACE) inhibitory activity was also investigated. Microginin 527 (4) was the most potent of the group, with an IC50 of 31 μM.

During an investigation of new actinomycete species from Caribbean sponges for novel bioactive natural products, frigocyclinone (1), dimethyldehydrorabelomycin (3) and six new angucyclinone derivatives were isolated from Streptomyces sp. strain M7_15 associated with the sponge Scopalina ruetzleri. Of these, monacyclinones A–B (4–5) contain the core ring structure of dehydrorabelomycin (2) with the aminodeoxysugar found in frigocyclinone (1). Monacyclinone C (6) is a hydroxylated variant of frigocyclinone (1) and monacyclinone D (7) is a Baeyer Villiger derivative of (6) which also exists as the open chain hydrolysis product monacyclinone E (8). Monacyclinone F (9) contains two unique epoxide rings attached to the angucyclinone moiety and an additional aminodeoxysugar attached through an angular oxygen bond. All structures were confirmed through spectral analyses. Activity against rhabdomycosarcoma cancer cells (SJCRH30) after 48 h of treatment was observed with frigocyclinone (1, EC50 = 5.2 µM), monacyclinone C (6, 160 µM), monacyclinone E (8, 270 µM), and monacyclinone F (9, 0.73 µM). The strongest bioactivity against rhabdomycosarcoma cancer cells and gram-positive bacteria was exhibited by compound 9, suggesting that the extra aminodeoxysugar subunit is important for biological activity.

Chemical investigation of a cultured strain of Microcystis aeruginosa revealed the presence of three new microginin analogs, microginins 680, 646, and 612. While nearly all other previously reported microginins possess the unusual 3-amino-2-hydroxydecanoic (Ahda) acyl moiety, these new compounds are members of an extremely rare sub-class containing the β-amino acid residue 3-amino-2-hydroxy-octanoic acid (Ahoa). Additionally, microginins 680 and 646 (di-chloro and mono-chloro, respectively) represent the first halogenated examples of the Ahoa group in a natural product.

The global need for accurate and sensitive quantitation of microcystins (MCs) persists as incidents of cyanobacterial harmful algal blooms continue to rise and recent research reveals an underestimation of the human health implications of these toxins. An optimal approach for their accurate quantitation relies on the availability of stable isotope-labeled MC standards for use in stable isotope dilution analysis (SIDA) strategies involving liquid chromatography tandem mass spectrometry (LC-MS/MS). Due to the dearth of isotopically labeled MCs, ten different

As environmental changes such as eutrophication lead to increased size and frequency of cyanobacterial blooms, research into the toxins produced by these blooms becomes increasingly important. One of the common toxins produced by cyanobacterial blooms is cylindrospermopsin ( 1 ), a potent inhibitor of protein synthesis. To date, only two additional analogs of cylindrospermopsin have been isolated, namely 7-epicylindrospermopsin ( 2 ) and 7-deoxy-cylindrospermopsin ( 3 ). This report details the isolation and structure determination of an additional two new analogs, 7-deoxy-desulfo-cylindrospermopsin ( 4 ) and 7-deoxy-desulfo-12-acetylcylindrospermopsin ( 5 ). These are the first new analogs of cylindrospermopsin to be reported in over a decade. Based on their structural features, it is likely that these new analogs also possess the harmful biological activities displayed by the rest of the cylindrospermopsin family.

Marine actinomycetes provide a rich source of structurally unique and bioactive secondary metabolites. Numerous genera of marine actinomycetes have been isolated from marine sediments as well as several sponge species. In this study, 16 different species of Caribbean sponges were collected from four different locations in the coastal waters off Puerto Rico in order to examine diversity and bioactive metabolite production of marine actinomycetes in Caribbean sponges. Sediments were also collected from each location, in order to compare actinomycete communities between these two types of samples. A total of 180 actinomycetes were isolated and identified based on 16S rRNA gene analysis. Phylogenetic analysis revealed the presence of at least 14 new phylotypes belonging to the genera Micromonospora, Verruscosispora, Streptomyces, Salinospora, Solwaraspora, Microbacterium and Cellulosimicrobium. Seventy-eight of the isolates (19 from sediments and 59 from sponges) shared 100 % sequence identity with Micromonospora sp. R1. Despite having identical 16S rRNA sequences, the bioactivity of extracts and subsequent fractions generated from the fermentation of both sponge- and sediment-derived isolates identical to Micromonospora sp. R1 varied greatly, with a marked increase in antibiotic metabolite production in those isolates derived from sponges. These results indicate that the chemical profiles of isolates with high 16S rRNA sequence homology to known strains can be diverse and dependent on the source of isolation. In addition, seven previously reported dihydroquinones produced by five different Streptomyces strains have been purified and characterized from one Streptomyces sp. strain isolated in this study from the Caribbean sponge Agelas sceptrum.

Defined experimental regimes were used to determine the effects of nutrient limitation on the toxicity of Alexandrium peruvianum in batch culture. Subsamples for cell counts and spiroimine analysis at six day intervals were used to investigate the concentrations and composition of these compounds throughout growth. An erythrocyte lysis assay for hemolytic activity was performed on cell pellets and supernatants also collected every six days over the entire growth period from all treatments. From the data, growth rates, cellular spiroimine quotas and effective concentration-fifty (EC50s) for cellular and supernatant associated hemolytic activity were calculated. Phosphate limitation was identified as a key regulator of toxicity in this species, yielding maximum values of 54.1 pg cell−1 for 13-desmethyl spirolide C, 96.4 pg cell−1 for 12-methylgymnodimine and a potent hemolytic EC50 value of 7.1 × 103 cells. The concentrations of spiroimines detected in A. peruvianum among various treatments, in addition to a unique profile of paralytic shellfish poisoning toxins, is unique in the body of microalgal literature. Because of the multiple toxin arsenal produced by this organism, the evaluation of a single toxin clearly would have underestimated the potential virulence and significance of this clone. This study provides the first evidence that growth and toxin production of A. peruvianum are influenced by altered nutrient ratios.

Phytoplankton monitoring in Wickford Cove, Rhode Island, US (41°34′10.13″N, 71°26′45.76″W), located in Narragansett Bay, detected an unusual species of Alexandrium in the spring of 2009. Thecal plate analysis using brightfield and SEM microscopy revealed a plate morphology consistent with that of Alexandrium peruvianum (Balech and de Mendiola) Balech and Tangen. Molecular analyses indicated that the sequences of the SSU, ITS1, 5.8S, ITS2 and LSU through the D region of the 18S gene were similar to those of A. peruvianum from North Carolina. Toxin analyses of cells brought into culture revealed saxitoxins, gymnodimine and fast-acting spiroimines were present in the cultured clone. Saxitoxins detected included GTX 2, GTX3, B1, STX, C1 and C2. Also present in the Wickford cove isolates of A. peruvianum were 12-methyl gymnodimine and 13-desmethyl spirolide C. A. peruvianum was detected at four sites in lower Narragansett Bay: at two sites in Wickford and two sites in Jamestown, RI. A. peruvianum was observed in the spring of 2009, 2010, 2011 and 2012 at maximum abundance levels ranging from tens of cells per liter to 14,000 cells L −1 . The discovery of A. peruvianum in Rhode Island coastal waters, with its potential threat to public health, is notable as it appears to be an emergent bloom species globally. The presence of A. peruvianum in Narragansett Bay is the third confirmed observation of this species on the Atlantic coast of North America. Monitoring efforts in the southern New England region should incorporate measures to detect the presence of A. peruvianum toxins.

Routine sampling of the water quality stations in the New River Estuary (Jacksonville, North Carolina, USA) during November 2004 revealed the presence of a previously unidentified dinoflagellate. Preliminary observations of its morphology suggested it to be consistent with that of Alexandrium peruvianum (Balech et Mendiola) Balech et Tangen. Observations using brightfield, epifluorescence and scanning electron microscopy confirmed the diagnostic thecal plates to be those of A. peruvanium . Clonal cultures established from cells isolated from the New River Estuary samples were also used for further studies of morphology and for the presence of toxins. Thecal morphology was consistent with that described by Balech clearly separating it from the sister species Alexandrium ostenfeldii . Three classes of toxins were detected from these cultures. An erythrocyte lysis assay (ELA) was used to confirm the presence of hemolytic toxins in A. peruvianum cultures. A cellular EC 50 for lysis was 1.418 × 10 4 cells, well within the range the maximal cells densities found in the New River and more potent when compared on a cellular basis with Prymnesium parvum . Another toxin class detected in A. peruvianum cultures was the fast acting 13-desmethy C and D spirolides also produced by the sister species A. ostenfeldii . The last toxin type detected in the A. peruvianum cultures was the paralytic shellfish toxins, GTX 2, 3, B1, STX and C1,2. These findings expand the geographic range of occurrence for A. peruvianum in the U.S. to be much greater than previously considered. The morphological characters agreed with previously reported molecular data in separating A. peruvianum from A. ostenfeldii . It is also the first confirmed report that this species produces PSP toxins, spirolides and naturally occurring hemolytic substances. In light of these findings additional attention is needed for the detection of Alexandrium species in all coastal waters of the U.S. This added effort will enhance the evaluation of the relative impacts of the species to shellfish safety and bloom surveillance.

The novel gymnodimine congener 12-methylgymnodimine was isolated from the dinoflagellate Alexandrium peruvianum from the New River in North Carolina. In addition, the presence of 13-desmethylspirolide C was confirmed by NMR characterization. This marks the first time these two classes of cyclic imines have been found in the same organism, providing further evidence for a direct genetic relationship between the biosynthetic pathways for gymnodimines and spirolides suggested by comparison of their structures.

The photodegradation rate of the brevetoxin analog PbTx-2 was investigated in a variety of natural water matrices. The observed first-order photodegradation rate coefficient of the logarithmic-transformed dissolved PbTx-2 concentrations vs. irradiation time was 0.20 ± 0.04 h−1 in coastal seawater, corresponding to a half-life of approximately 3 h based on 10 separate photolysis experiments. No loss of PbTx-2 occurred in dark controls, indicating that this was primarily a photo-mediated process. Photodegradation rate coefficients in samples in which dissolved organic matter was removed by ultraviolet oxidation prior to photolysis resulted in significantly slower rates of PbTx-2 photodegradation (0.08 ± 0.03 h−1). When trace metals were also removed prior to photolysis, no loss of PbTx-2 occurred, indicating that direct photolytic loss of PbTx-2 is insignificant in coastal seawater. The proposed mechanism of PbTx-2 decay is a photosensitized pathway involving organic matter and trace metals that is significantly accelerated by decreasing oxygen concentrations. The influence of molecular oxygen on the rate of toxin loss has important ramifications for the fate of PbTx-2 during Karenia brevis blooms, as in situ dissolved O2 concentrations fluctuate widely during bloom development and decay. Sunlight-mediated reactions are therefore a significant, yet previously unrecognized, sink of dissolved PbTx-2 in seawater under environmentally relevant conditions.

Synthesis of the BC/DE ring model of brevisin, a polycyclic ether isolated from the red tide dinoflagellate Karenia brevis, is reported. Comparison of the NMR data of the BC/DE ring model with those corresponding to the same region of brevisin led to the confirmation of its structure around the acyclic juncture.

An improved synthesis of (−)-brevisamide a marine cyclic ether isolated from the red-tide dinoflagellate Karenia brevis was achieved. The ether ring portion was constructed from an unsaturated lactone, which was prepared enantioselectively via an Evans aldol reaction and one-pot lactonization in the presence of excessive base after an Ando reaction. The ether ring and a dienol side chain fragment were connected via Suzuki–Miyaura coupling.

The dinoflagellate Karenia brevis is known for the production of brevetoxins, a family of polycyclic ether toxins, as well as their antagonist brevenal. Further examination of organic extracts of K. brevis has uncovered yet another unprecedented cyclic ether alkaloid named brevisamide. This report describes the structure elucidation of brevisamide based on detailed MS and NMR spectral analysis, and the importance of this new compound in shedding light on the biogenesis of fused polyethers is discussed.

The photochemistry of the marine toxin domoic acid (DA) and its related compounds in aqueous solution was investigated under environmentally relevant concentrations and radiation conditions. DA was readily phototransformed under simulated sunlight, showing exponential decay as a function of irradiation time. Major identifiable intermediate products were three geometric isomers identified as isodomoic acids D, E, and F. Isodomoic acids D and E formed much more quickly and in higher concentrations relative to isodomoic acid F when DA was irradiated. When pure solutions of either isodomoic acids D, E, or F were irradiated, there was concomitant production of the two other isomers as well as production of DA. These results are significant because they indicate that the three isomers produced upon photolysis of DA are also photolabile and are capable of regenerating the original form of the toxin. A group of less polar products all with a molecular weight of 267 Da was also detected in irradiated solutions of DA. These products are tentatively identified as decarboxylated derivatives of DA. These results demonstrate that exposure of DA to simulated sunlight modifies the chemical structure of DA and produces a suite of isomers and decarboxylated derivatives that can potentially affect the toxicity and metal binding capacity of the original toxin. It is also shown that the photochemistry of DA is a complex system involving simultaneous reversible (isomerization) and irreversible (e.g. decarboxylation) reactions. Therefore, results from this study suggest that isodomoic acids D, E, or F might act as photo-intermediates in the photochemical degradation of DA in the ocean.

The biogenetic origin of the carbon atoms in the 2-azaanthraquinone scorpinone ( 1), produced by the rare fungus Amorosia littoralis isolated from marine sediment, was explored through isotopic enrichment studies utilizing [2- (13)C]-acetate and [1,2- (13)C]-acetate. The labeling results reveal a heptaketide precursor is involved in the biosynthesis of 1, as has been found for the structurally related naphthoquinone dihydrofusarubin. The previously identified naphthoquinone herbarin ( 2) was also isolated and appears to bear the same biogenetic relationship to 1 as the fusarubins do to the fungal 2-azaanthraquinone bostrycoidins.

The adsorption of dissolved domoic acid (DA) and its geometrical isomers was assessed in aqueous solutions containing various types of particles. In one series of experiments carried out in coastal seawater, detectable net adsorption of 100 nM DA occurred only onto natural seawater particles (unfiltered seawater) and 0.5 g L −1 chromatographic silica (18%) in 0.2 μm-filtered seawater. Some net adsorption ( −1 suspension of estuarine sediment and 0.5 g L −1 solution of humic acid in filtered seawater. No losses were seen in 0.5 g L −1 suspensions of illite, kaolinite, montmorillonite, and silica sand. Biological degradation accounted for small losses (8–10%) in filtered seawater without particles. A second series of experiments using organic-free, iso -DA D, E, and F) showed negligible adsorption (0–8%) onto a variety of particles in filtered seawater, suggesting that major ions in seawater neutralize electrostatic attractions between particles and DA isomers. These results suggest that DA and its isomers are relatively hydrophilic and not particle reactive. Our data suggest that photochemical and biological degradation of dissolved DA and its isomers appears to occur in bulk surface seawater and its transport to bottom sediments must be mainly biologically driven.

Understanding the biosynthesis of dinoflagellate polyketides presents many unique challenges. Because of the remaining hurdles to dinoflagellate genome sequencing, precursor labeling studies remain the only viable way to investigate dinoflagellate biosynthesis. However, prior studies have shown that polyketide chain assembly does not follow any of the established processes. Additionally, acetate, the common precursor for polyketides, is frequently scrambled, thus compromising interpretation. These factors are further compounded by low production yields of the compounds of interest. A recent report on the biosynthesis of spirolides, a group belonging to the growing class of toxic spiroimines, provided some insight into the polyketide assembly process based on acetate labeling studies, but many details were left uncertain. By feeding (13)C methyl-labeled methionine to cultures of Alexandrium ostenfeldii, the producing organism of 13-desmethylspirolide C, and application of the odd-even methylation rule, the complete biosynthetic pathway has been established.

The endangered seagrass Halophila johnsonii Eiseman, exhibits high-light adapted photophysiology consistent with its distribution in intertidal and shallow subtidal (0–3 m) coastal-lagoon habitats along 200 km of southeastern Florida. To examine the short-term responses of this seagrass to three controlled-irradiance treatments (PAR + UVA + UVB [full spectrum], PAR + UVA, and PAR only), greenhouse-acclimated plants were transferred to outdoor mesocosms during July–August 2002. Chlorophyll fluorescence, UV fluorescence, and samples for pigment extraction were collected in the greenhouse, prior to moving the plants outside and on days 1, 2, 3, 4, 6, 10, and 21 of the 24-day experiment. Typical of sun-adapted plants, effective quantum yields measured by pulse-amplitude modulated (PAM) fluorometry were relatively low in all treatments, ranging from 0.46 ± 0.09 (PAR only) to 0.58 ± 0.08 (PAR + UVA + UVB). In the PAR only treatments, there were strong effects on days 1 and 4, presumably because the irradiance in the greenhouse not only lacked all λ

This study was designed to investigate the lipid-lowering ability of a novel dietary ingredient composed of phytosterols esterified to (n-3) polyunsaturated fatty acids (PUFA) [PS(n-3)]. Adult guinea pigs were fed a test diet supplemented with PS(n-3) (25 g/kg) and corn oil (CO, 5 g/kg), whereas the diet fed to control guinea pigs was supplemented with CO only (30 g/kg). Cholesterol was added to both diets (0.8 g/kg). After 3-4 wk of consuming the diets, serum total cholesterol (TC) and triacylglycerol (TAG) in the PS(n-3) group were 36 and 29% lower, respectively, than levels in controls (P0.05). The lower TC levels in the PS(n-3) group reflected a 38% reduction in non-HDL cholesterol (non-HDL-C), whereas the HDL-C concentration was unaffected. Analysis of cardiac left ventricle indicated that generation of the proaggregatory, arrhythmic eicosanoid, thromboxane A(2), was60% lower in the PS(n-3)-supplemented guinea pigs than in CO controls (P0.001). This study demonstrates that the TAG-lowering and eicosanoid-modifying properties of the fish oil (n-3) PUFA are retained when they are provided in the diet in ester linkage with hypocholesterolemic phytosterols.

The syndrome that is characterized by obesity, insulin resistance, and hyperlipidemia is increasingly prevalent in all prosperous societies. It is now recognized as a major contributor to cardiovascular disease. Vascular dysfunction in the form of hypercontractility and impaired nitric oxide-mediated relaxation is a significant component of cardiovascular disease, predisposing to ischemic events. The JCR:LA-cp strain of rats exhibits all major aspects of the obesity/insulin resistance syndrome, including vascular dysfunction and ischemic lesions of the heart. Dietary lipid intake may have a marked effect on plasma lipid levels and, potentially, on vascular disease. We have investigated the effects of a novel preparation, ONC101 (a phytosterol esterified with fish oil), on plasma lipids and vascular function in the insulin-resistant JCR:LA-cp rat. Treatment of obese male rats with ONC101 from 8 to 12 wk of age resulted in no change in plasma lipid concentrations at 0.5 g/kg body weight. At the higher dose of 2.6 g/kg, plasma TG fell 50% (1.26 vs. 2.59 mmol/L, P

The relative stereochemistry of 13-desmethyl spirolide C, except for one chiral center, has been determined from NMR data by means of ConGen, a molecular modeling method which applies high-temperature molecular dynamics under distance constraints generated from NOESY and ROESY data. The method shows this spirolide to have the same relative stereochemistry as pinnatoxins A and D in the region of their common structure. Applicability of the ConGen method to molecules of this type is further justified by demonstrating that it yields the correct relative stereochemistry of the pinnatoxins when used with constraints generated from published data. The relative stereochemistries of spirolides B and D are also determined by comparisons of their NMR data with 13-desmethyl spirolide C and further application of ConGen.

The discovery of brevisin, the first example of an “interrupted” polycyclic ether, obtained from the dinoflagellate Karenia brevis, posed some important questions regarding the mechanism of the cyclization process. Consequently, we have established absolute configurations of brevisin and its related metabolite brevisamide using a modified Mosher's esterification method. For brevisin, analysis was carried out on both the 31-monokis- and the 10,31-bis-MTPA esters. The results suggest that both metabolites, like other polyethers from K. brevis, result from polyepoxide precursors with uniform (S, S) configurations for all epoxides, and provide further support for a universal stereochemical model for dinoflagellate polyether formation.

A monocyclic ether alkaloid, brevisamide ( 1 ) was isolated from the dinoflagellate Karenia brevis that produces a variety of ladder-frame polyethers. Its proposed biosynthetic intermediate ( 2 ) comprising a linear backbone with an E -olefin functionality was synthesized for biosynthetic studies on the marine ladder-frame polyethers.

Hoffmaniolide, a novel macrolide, was isolated and identified from the marine dinoflagellate Prorocentrum hoffmannianum .

The cross-reactivity of the 6/50 monoclonal anti-okadaic acid antibody (mAb) to the recently discovered diarrhetic shellfish poisoning (DSP) metabolites dinophysistoxin-4 (DTX-4), dinophysistoxin-5 (DTX-5), and an okadaic acid (OA) diol ester was determined using a competitive indirect enzyme-linked immunosorbent assay (ELISA). The reactivity of the antibody to these molecules was compared to that with OA; the 6/50 mAb recognized all of these DSP compounds with equal sensitivity within the working range of the antibody (10–100 nM for OA). This confirms the ability of the antibody to detect all DSP compounds when used in analyses including ELISA and immunocytochemistry.

The biosynthesis of the neurotoxin domoic acid (DA) in the diatom Pseudo-nitzschia multiseries was investigated using 13C- and 14C-labelled precursors. The labelling pattern determined by NMR spectroscopy following incorporation of [1,2-13C2]-acetate showed enrichment of every carbon of DA. The enrichment levels were consistent with a biosynthetic pathway involving two different intermediate precursor units. Addition of labelled acetate either early or late during exponential growth gave similar patterns and levels of incorporation. Analysis of the labelling pattern indicated that DA is biosynthesised by condensation of an isoprenoid intermediate with another intermediate derived from the tricarboxylic acid (TCA) cycle. The absence of deuterium at C2 in DA following incorporation of [2-13C, 2H3]-acetate is consistent with α-ketoglutarate or a derivative as the TCA cycle-derived intermediate. The different incorporation efficiencies of acetate into the putative precursor intermediates suggest that either each unit is biosynthesized in a different part of the diatom cell, or that the isoprene chain is not assembled by the usual acetate–mevalonate pathway. The latter proposal is supported by the complete absence of deuterium retention in the isoprenoid-derived portion following incorporation of [2-13C, 2H3]-acetate. Copyright © 1998 John Wiley & Sons, Ltd.

A thin-layer chromatography (TLC) method has been developed for the semi-quantitative analysis of domoic acid (DA) in shellfish tissues. Tissues were extracted in a single-step homogenization of tissue with 50 % aqueous methanol and then taken through a selective strong anion exchange cleanup. Cleaned extracts were applied directly to silica gel TLC plates and developed with a butanol–acetic acid–water mixture (3:1:1, Rf = 0.45 for DA). As little as 10 µg DA per gram of tissue could be detected after chromatography using a hand-held short-wave UV lamp to detect fluorescence quenching. Confirmation was provided by spraying the plate with ninhydrin, which reacts with the secondary amine of DA to give a distinctive yellow colored product. The extraction, cleanup and TLC procedures are fast and simple, and do not require the use of expensive equipment. This method should prove useful for the routine screening of shellfish tissues in those laboratories not equipped with an LC system. It should also be useful as a chemical confirmation method for DA in samples tested positive by assay methods such as immunoassay. Copyright © 1999 John Wiley & Sons, Ltd.

Pectenotoxins (PTXs) isolated from the scallop Patinopecten yessoensis were shown to be involved in an episode of diarrhetic shellfish poisoning (DSP). A total of eight analogues (PTX1-PTX7 and PTX10) have been isolated to date, and the structures of four of these analogues (PTX1, PTX2, PTX3, and PTX6) have already been elucidated. Here, we report the characterization of PTX4 and PTX7 as 7-epi-PTX1 and 7-epi-PTX6, respectively, on the basis of NMR data and an acid-catalyzed chemical interconversion. The structures of two new artifacts, PTX8 and PTX9, produced following this treatment are also reported.

Four new tricarbocyclic diterpenes 2–5 containing the bicyclic [4.3.1] ring system found in floridicin (1), and an additional four dicarbocylic terpenes 6–9 containing the same bicyclic moiety, have been isolated from the soft coral, Xenia florida. Their structures were elecidated by spectroscopic means.

The Cape Fear River is the largest river system in North Carolina. It is heavily used as a source of drinking water for humans and livestock as well as a source of irrigation water for crops, and production water for industry. It also serves as a major fishery for both commercial and recreational use. In recent years, possibly related to increased eutrophication of the river, massive blooms of cyanobacteria, identified as Microcystis aeruginosa have been observed. Bloom samples collected in 2009 and 2012 were chemically analyzed to determine if they contained cyanobacterial toxins known as microcystins. Both blooms were found to produce microcystins in high yields. Microcystins are potent hepatotoxins that can be bio-accumulated in the food chain. Recent biological studies have also shown a host of other potentially harmful effects of low level microcystin exposure. Detailed chemical analysis of these blooms led us to discover that these blooms produce an additional family of cyanobacterial peptides know as the micropeptins, including two new members named micropeptins 1106 and 1120. The biological activities of these new molecules have not yet been determined, although protease activity has been well documented for this peptide group. These data indicate a need for thorough monitoring of toxin levels especially during bloom events in addition to additional biological testing of other cyanopeptides present in blooms.

Through a combination of chemical and molecular analysis, a new polyene macrolactam named micromonolactam was obtained from two marine-derived Micromonospora species. This new polyene metabolite is a constitutional isomer of salinilactam A but contains a different polyene pattern and one cis double bond, in contrast to the all trans structure reported for salinilactam A. The molecular analysis data also established that micromonolactam is a hybrid polyketide derived from 11 polyketide units and a modified glutamate starter unit.

Two new spirolide derivatives, E and F, have been isolated in low yield from shellfish extracts. Absence of activity in the mouse bioassay of these derivatives, and of the secondary amine reduction product of spirolide B, identifies the spirolide pharmacophore as the cyclic imine moiety.

Two new metabolites (1 and 2) were isolated from the marine fungus Microsphaeropsis olivacea. The structures were elucidated, through analysis of the spectroscopic data, as an unusual methyl-branched unsaturated fatty acid, 10-methyl-9Z-octadecenoic acid (1), and the glyceride (2). The locations of the double bond and the methyl branching were determined from the electron impact (EI) mass spectrum of the picolinyl derivative of 1 and from MS/MS data on the hydrogenation products of 1 and 2. Key words: Microsphaeropsis olivacea, marine fungus, methyl-branched unsaturated fatty acid, picolinyl ester.

The diarrhetic shellfish poisoning toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1) are potent phosphatase inhibitors produced by certain species of marine dinoflagellates. OA can cause hyperphosphorylation of a broad range of animal and higherpalnt proteins, but little is known regarding the effects of the DSP toxins on marine organisms or their biological function. A variety of microalgae, including a clone ofProrocentrum lima known to produce both OA and DTX-1, were incubated with solutions of OA and in one case DTX-1 or a combination of OA and DTX-1. OA inhibited the growth of all non-DSP-producing test species at micromolar concentrations, butP. lima was not affected even at much higher levels. This differential activity of OA suggests that the DSP toxins may play an allelopathic role and raises questions regarding the strategies adopted by DSP-producing dinoflagellates such asP. lima to avoid autotoxicity. The effects of DTX-1 on microalgal growth were found to be equivalentt to those of OA, and the effects of both toxins in combination were simply additive.

A new toxin, prorocentrolide B (1), has been isolated following bioassay-guided fractionation of a BuOH extract of the tropical dinoflagellate, Prorocentrum maculosum Faust. This compound produces a rapid toxic response in the mouse bioassay, a type of activity not accounted for by other diarrhetic shellfish poisoning toxins produced by P. maculosum. The structure 1 was established by NMR and MS and is similar to prorocentrolide (2), a toxin from a strain of Prorocentrum lima. NMR data and the modeling program ConGen have been used to establish the relative stereochemistry of some individual ether ring systems and the hexahydroisoquinoline ring.

Two novel water-soluble sulfated DSP toxin derivatives 4 and 5, are reported from the benthic dinoflagellate Prorocentrum maculosum. Their occurrence supports the idea that all DSP toxin-producing Prorocentrum species biosynthesize such compounds as a means of toxin storage, and perhaps as a means of eventually exporting them from the cell.

A new member of the gymnodimine class of spiroimine toxins has been isolated from a laboratory culture strain of Alexandrium ostenfeldii. Extensive one-dimensional (1D) and two-dimensional (2D) NMR data analysis was used to elucidate its structure as 12-methylgymnodimine B.

In most maritime countries, the seafood industry is an important source of revenue and accounts for a significant portion of the country's food supply. However, certain seafood, in particular shellfish, may become contaminated with naturally occurring toxins. The existence of shellfish toxins has been known for centuries and early attempts to deal with this threat led to the creation of folklore rituals and religious customs among tribal and ethnic groups. Eventually it was found that these toxins are produced by microbial organisms, usually phytoplankton, that occur naturally in the ocean. In Canada, as well as other countries with cold or temperate coastal waters, three groups of toxins are of particular importance and these are the paralytic shellfish poisoning (PSP) toxins, the diarrhetic shellfish poisoning (DSP) toxins and amnesic shellfish poisoning (ASP) toxin. Since it is virtually impossible to prevent the occurrence of these toxins, the approach in modern times has been to monitor seafood for the presence or absence of toxins using a standardized mouse bioassay. However, not all toxins can be detected effectively by this method and there is a trend in several countries to dispense with the mouse bioassay. Furthermore, there are differences between countries as to the preferred method of sampling and analysis and there is not agreement between all countries concerning tolerance levels of the various toxins in seafood. These difficulties, the occurrence of new toxins, and the rapid expansion of the shellfish aquaculture industry have put considerable pressure upon the scientific community to develop new methods of coping with these toxins, including more sensitive chemical or biochemical assays that are rapid and inexpensive. The possibilities and options for coping with these ubiquitous toxins are presented and discussed.

Five new compounds related to the neurotoxin domoic acid have been characterized by spectroscopic methods. They include a diastereoisomer that occurs naturally with domoic acid in mussels and four hydrogenation products. The stereochemistry of the two epimeric tetrahydro derivatives could not be assigned on the basis of the NMR data alone because of high conformational flexibility. However, a clear-cut assignment was possible by molecular modelling in combination with the NMR data.

The first total synthesis of (I) succeeds in 29 steps from commercially available deoxy-D-ribose.

The first and highly efficient total synthesis of (−)-brevisin has been achieved. The title compound was synthesized in only 29 steps (longest linear sequence) from commercially available starting materials. The synthesis provided over 70 mg of a marine polycyclic ether compound.